Figure 3. STAT3-deficient memory B cells differentiate into Ab-secreting cells in response to IL-21. (A–D) Naive (CD20+CD10−CD27−IgG−) and memory (CD20+CD10-CD27+) B cells were sort-purified from normal donors (A and B, n = 16; C and D, n = 7), STAT3MUT patients (A and B, n = 8; C and D, n = 7), or STAT1MUT patients (n = 6), and then cultured with CD40L alone or together with IL-21 (A and B) or CpG (C and D). The levels of secreted IgM, IgG, and IgA were determined by ELISA after 10–12 d. The columns represent the mean ± SEM of experiments performed using naive B cells from 7–16 normal donors, 7–8 STAT3MUT patients, or 6 STAT1MUT patients. (E and F) Naive and memory B cells were sort-purified from normal donors or STAT3MUT patients and then cultured with CD40L alone or together with IL-4. Expression of AICDA (E) and Ig ε germline transcript (GLT; F) was determined by qPCR and PCR, respectively. The graphs in E represent the mean ± SEM of three experiments using B cells from different donors or patients. The gel depicted in F is representative of experiments performed using B cells from two to three different donors or patients. (G and H) Naive and memory B cells were sort-purified from a single normal donor or STAT3MUT patient and then cultured with CD40L alone or together with IL-21 or CpG. Proliferation was assessed after 5 d by determining incorporation of [3H]thymidine during the last 18 h of culture. The graphs are the mean ± SEM of replicate cultures of naive or memory B cells from one normal donor or one STAT3MUT patient. The annotated values indicate the fold increase in proliferation of normal or STAT3MUT naive or memory B cells cultured with CD40L/IL-21 or CD40L/CpG over that induced by CD40L alone. (I and J) Normal naive (N) or normal, STAT3MUT, or STAT1MUT memory (M) B cells (n = 6; I) or total PBMCs from normal donors or STAT3MUT patients (n = 4; J) were cultured with CD40L and IL-21 for 10–12 d. The levels of antitetanus IgG in culture supernatants were determined by ELISA using immobilized tetanus toxoid as solid phase Ag. Each symbol represents the response of B cells from an individual control or patient; the horizontal bars represent means. ns, no significant; *, P < 0.05; **, P < 0.01.
Figure 3.

STAT3-deficient memory B cells differentiate into Ab-secreting cells in response to IL-21. (A–D) Naive (CD20+CD10−CD27−IgG−) and memory (CD20+CD10-CD27+) B cells were sort-purified from normal donors (A and B, n = 16; C and D, n = 7), STAT3MUT patients (A and B, n = 8; C and D, n = 7), or STAT1MUT patients (n = 6), and then cultured with CD40L alone or together with IL-21 (A and B) or CpG (C and D). The levels of secreted IgM, IgG, and IgA were determined by ELISA after 10–12 d. The columns represent the mean ± SEM of experiments performed using naive B cells from 7–16 normal donors, 7–8 STAT3MUT patients, or 6 STAT1MUT patients. (E and F) Naive and memory B cells were sort-purified from normal donors or STAT3MUT patients and then cultured with CD40L alone or together with IL-4. Expression of AICDA (E) and Ig ε germline transcript (GLT; F) was determined by qPCR and PCR, respectively. The graphs in E represent the mean ± SEM of three experiments using B cells from different donors or patients. The gel depicted in F is representative of experiments performed using B cells from two to three different donors or patients. (G and H) Naive and memory B cells were sort-purified from a single normal donor or STAT3MUT patient and then cultured with CD40L alone or together with IL-21 or CpG. Proliferation was assessed after 5 d by determining incorporation of [3H]thymidine during the last 18 h of culture. The graphs are the mean ± SEM of replicate cultures of naive or memory B cells from one normal donor or one STAT3MUT patient. The annotated values indicate the fold increase in proliferation of normal or STAT3MUT naive or memory B cells cultured with CD40L/IL-21 or CD40L/CpG over that induced by CD40L alone. (I and J) Normal naive (N) or normal, STAT3MUT, or STAT1MUT memory (M) B cells (n = 6; I) or total PBMCs from normal donors or STAT3MUT patients (n = 4; J) were cultured with CD40L and IL-21 for 10–12 d. The levels of antitetanus IgG in culture supernatants were determined by ELISA using immobilized tetanus toxoid as solid phase Ag. Each symbol represents the response of B cells from an individual control or patient; the horizontal bars represent means. ns, no significant; *, P < 0.05; **, P < 0.01.

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